Anti-apoptotic BCL-2 family members regulate hematopoiesis and when dysregulated contribute to cancer, immunodeficiency, and autoimmunity. MCL-1 is absolutely required for cell survival at multiple stages of hematopoietic development. In contrast, genetic ablation of Bcl-2 or Bcl-X has revealed specific roles in promoting hematopoietic survival. Why MCL-1 plays such a critical role in maintaining survival and why the concomitant endogenous expression of other anti-apoptotic regulators cannot compensate for MCL-1 loss has remained unresolved. Therefore, the long term goal of my laboratory is to understand how MCL-1 functions and is regulated during hematopoietic development and survival. To understand the requirement for MCL-1 in development, we have recently identified that it localizes to both the outer mitochondrial membrane, where it MCL-1 binds and sequesters pro-apoptotic molecules, and to the mitochondrial inner membrane as an N- terminally truncated form. The contribution of both of these localizations to MCL-1's function is unclear. The objective of this proposal is to dissect the functional roles of the two mitochondrial forms of MCL-1 and to assess the individual roles of these localizations in promoting cellular survival. Our central hypothesis is that the outer membrane form of MCL-1 acts like "classical" BCL-2 family members to antagonize pro-apoptotics, while the inner membrane form of MCL-1 regulates cell survival by modulating mitochondrial function. The studies we propose will reveal a previously unrecognized role for MCL-1 and shed light onto a new mechanism by which an anti-apoptotic BCL-2 family member promotes mitochondrial function. Aim 1: Define the role(s) for the different mitochondrial forms of MCL-1 in regulating hematopoiesis. MCL-1 exhibits a profound requirement during many stages of hematopoiesis, but it is unclear why. Aim 2: Define the role(s) of inner mitochondrial MCL-1 in regulating apoptosis. Our preliminary studies indicate that only the outer mitochondrial form of MCL-1 is capable of binding and sequestering pro-apoptotic molecules. However, it is unclear whether the inner mitochondrial form of MCL-1 may alter response to death stimuli. Therefore, we will investigate how the different forms of MCL-1 contribute to its "classical" anti-apoptotic nature. Aim 3: Identify how MCL-1 regulates mitochondrial physiology. Our preliminary studies indicate that Mcl-1-deficiency results in abnormalities in mitochondrial physiology even when cells are not undergoing apoptosis. My laboratory has made many of the seminal findings defining the role of MCL-1 in promoting survival during hematopoiesis. Furthermore, we are uniquely positioned to successfully perform these studies as we have identified the novel localization, generated MCL-1 mutants that can dissect the different forms of MCL-1, and have extensive experience in MCL-1 biology. At the end of this study, we will have illuminated a previously unrecognized role for MCL-1 in promoting mitochondrial function. PUBLIC HEALTH RELEVANCE: Defects in the balance of cell survival and death can lead to a variety of human diseases including autoimmunity, immunodeficiency, and cancer. MCL-1 is a critical regulator of blood cell survival whose expression is tightly controlled in normal cells, but in cancerous cells such control is lost leading to inappropriate survival. This proposal is relevant to the goals of the National Institutes of Health because it investigates a novel role for MCL-1 in normal blood cells and will assess how MCL-1 can render cancer cells resistant to death.